Long non coding RNA ANRIL promotes non small cell lung ......Dec 27, 2014 · 1 Long non-coding RNA ANRIL promotes non small cell lung cancer cells proliferation and . inhibits. apoptosis

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Long non-coding RNA ANRIL promotes non small cell lung cancer cells proliferation and

inhibits apoptosis by silencing KLF2 and P21 expression

Feng-qi Nie1#

, Ming Sun2#

, Jin-song Yang3#

, Min Xie2, Tong-peng Xu

1, Rui Xia

2, Yan-wen

Liu2, Xiang-hua Liu

2, Er-bao Zhang

2, Kai-hua Lu

1* and Yong-qian Shu

1*

1Department of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing,

People’s Republic; 2Department of Biochemistry and Molecular Biology, Nanjing Medical

University, Nanjing, People’s Republic of China;3Department of Oncology, Nanjing First

Hospital, Nanjing Medical University, P. R. China.

*Corresponding author:

Kai-hua Lu, Department of Oncology, First Affiliated Hospital, Nanjing Medical University,

Nanjing, People’s Republic,Tel:+86-025 68136711 ; Fax: +86-025 68136711; E-mail:

[email protected].

Yong-qian Shu, Department of Oncology, First Affiliated Hospital, Nanjing Medical

University, Nanjing, People’s Republic, E-mail: [email protected]

#These authors contributed equally to this work and should be regarded as joint first

authors

Ruining title: ANRIL promotes proliferation in NSCLC

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by grants from the National Natural Scientific Foundation of

China No. 81372397 to KH. Lu, 81301824 to XH. Liu, 81172140 and 81272532 to YQ Shu;

M. Sun was supported by a Jiangsu province ordinary university graduate student

research innovation project for 2013 (CXZZ13_0562, JX22013265).

on May 5, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 12, 2014; DOI: 10.1158/1535-7163.MCT-14-0492

mailto:[email protected]

http://mct.aacrjournals.org/

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Abstract

Recent evidence highlight long non-coding RNAs (lncRNAs) as crucial regulators of

cancer biology, which contribute to essential cancer cell functions such as cell proliferation,

apoptosis and metastasis. In non small cell lung cancer (NSCLC), several lncRNAs

expression are misregulated and have been nominated as critical actors in NSCLC

tumorigenesis. LncRNA ANRIL was firstly found to be required for the PRC2 recruitment

to and silencing of p15INK4B

, which expression is induced by ATM-E2F1 signaling pathway.

Our previous study showed that ANRIL was significantly up-regulated in gastric cancer,

and it could promote cell proliferation and inhibit cell apoptosis by silencing of miR-99a

and miR-449a transcription. However, its clinical significance and potential role in NSCLC

is still not documented. In this study, we reported that ANRIL expression was increased in

NSCLC tissues and Its expression level was significantly correlated with TNM stages and

tumor size. Moreover, patients with high levels of ANRIL expression had a relatively poor

prognosis. In addition, taking advantage of loss of function experiments in NSCLC cells,

we found that knockdown of ANRIL expression could impair cell proliferation and induce

cell apoptosis both in vitro and vivo. Furthermore, we uncover that ANRIL could not

repress p15 expression in PC9 cells, but through silencing of KLF2 and P21 transcription.

Thus, we conclusively demonstrate that lncRNA ANRIL plays an key role in NSCLC

development by associating its expression with survival in NSCLC patients, providing

novel insights on the function of lncRNA-driven tumorigenesis.

Key words: NSCLC; ANRIL; cell proliferation; KLF2; P21




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Introduction

Lung cancer is the most common type of cancer and the primary cause of cancer

death worldwide (1). NSCLC accounts for 80% of all lung cancer cases, represents the

most prevalent class of this cancer type and includes several histological subtypes such

as squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma (LCC) (2,

3). In spite of current advances in the treatments for NSCLC, including surgical therapy,

chemotherapy and molecular targeting therapy, the overall 5-year survival rate for NSCLC

patients have not been markedly improved over years and remains as low as 15% (4).

Therefore, a greater understanding of the molecular mechanisms involved in the

development, progression and spread of the NSCLC is essential for the developing of

specific diagnostic methods and designing of more individualized and effective

therapeutic strategies.

Recently, studies using the great advances in genomic technologies have revealed

the majority of the human genome is transcribed, whereas only 2% of the transcribed

genome codes for protein (5). Meanwhile, it is becoming increasingly apparent that the

large majority of genome is transcribed into non-coding RNAs (ncRNAs) including

microRNAs and long non-coding RNAs (lncRNAs) (6). The ENCODE Consortium has

elucidated the prevalence of thousands of human lncRNAs, but only very few of them

have been assigned with any biological function (7). To date, studies showed that miRNAs

play important roles in the post-transcriptional regulation of gene expression; however, the

lncRNAs counterpart of ncRNA is not well characterized (8). Although very few are

characterized in detail, LncRNAs are involved in a large range of biological processes,




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including modulation of apoptosis and invasion, reprogramming stem cell pluripotency,

and parental imprinting through the regulation of gene expression by chromatin

remodeling, histone protein modification, regulation of mRNA splicing and acting as

sponges for microRNAs (9-12).

In the past decade, lots of evidence have linked the dysregulation of lncRNAs with

diverse human diseases, in particular cancers (13-15). Therefore, identification of

cancer-associated lncRNAs and investigation of their molecular and biological functions

are important in understanding the molecular biology of NSCLC development and

progression. Our previous study showed that lncRNA ANRIL was significantly

up-regulated in gastric cancer, and increased ANRIL promoted gastric cancer cells

proliferation and inhibited apoptosis by epigenetic silencing of miR-99a and miR-449a

transcription (16). Moreover, ANRIL can bind to and recruits PRC2 to repress the

expression of p15INK4B locus, which resulted in increased cell proliferation (17, 18).

However, the ANRIL clinical significance and potential role in NSCLC development and

progression is still not documented.

In this study, we found that lncRNA ANRIL expression was increased in NSCLC

tissues compared with adjacent normal tissues. Its expression level was significantly

correlated with TNM stages and tumor size. Moreover, patients with higher level of ANRIL

expression had a relatively poor prognosis. Furthermore, we investigated the effects of

ANRIL expression on NSCLC cell phenotype in vitro and in vivo with loss of function study.

Moreover, we also showed that ANRIL could bind to PRC2 to repress KLF2 and P21

transcription, but not regulate P15INK4

expression in NSCLC PC9 cells, which indicated




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that ANRIL affected NSCLC cells proliferation and apoptosis partly via silencing of KLF2

and P21 transcription. This study advances our understanding of the role of lncRNAs,

such as a regulator of pathogenesis of NSCLC and facilitates the development of

lncRNA-directed diagnostics and therapeutics.

Materials and Methods

Tissue collection

We obtained 68 paired NSCLC and adjacent non-tumor lung tissues from patients who

underwent surgery at Jiangsu Province Hospital between 2010 and 2011, and were

diagnosed with NSCLC based on histopathological evaluation. Clinicopathological

characteristics, including tumor-node-metastasis (TNM) staging, were recorded. No local

or systemic treatment was conducted in these patients before surgery. All collected tissue

samples were immediately snap-frozen in liquid nitrogen and stored at –80°C until

required. Our study was approved by the Research Ethics Committee of Nanjing Medical

University, China. Written informed consent was obtained from all patients.

Cell lines

Five NSCLC adenocarcinoma cell lines (PC9, SPC-A1, NCI-H1975, H1299, and

H358), and one NSCLC squamous carcinomas cell lines (H520) were purchased from the

Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai,

China). A549, H1975, H1299 and H520 cells were cultured in RPMI 1640; 16HBE, PC9

and SPC-A1 cells were cultured in DMEM (GIBCO-BRL) medium supplemented with 10%

fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen,




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Carlsbad, CA, USA) at 37ºC/5% CO2. All cell lines were authenticated by short tandem

repeat DNA profiling.

RNA extraction and qPCR assays

Total RNA was isolated with Trizol reagent (Invitrogen) according to the

manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed in a final volume

of 10 µl using random primers under standard conditions for the PrimeScript RT reagent

Kit (TaKaRa, Dalian, China). We used the SYBR Premix Ex Taq (TaKaRa, Dalian, China)

to determine ANRIL expression levels, following the manufacturer’s instructions. Results

were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase

(GAPDH). The specific primers used are shown in Supplementary table S1. The qPCR

assays were conducted on an ABI 7500, and data collected with this instrument. Our

qPCR results were analyzed and expressed relative to threshold cycle (CT) values, and

then converted to fold changes.

Cell transfection

Human ANRIL cDNA clone L6ChoCKO-2-E10 with functional region was provided by

the Functional Genomics Research Center, KRIBB, Korea. Plasmid vectors

(pCNS-ANRIL, sh-ANRIL and empty vector) for transfection were prepared using DNA

Midiprep or Midiprep kits (Qiagen, Hilden, Germany), and transfected into16HBE, SPC-A1,

H1299 or PC9cells. The si-ANRIL or si-NC were transfected into SPC-A1, H1299 or PC9

cells. SPC-A1, H1299 or PC9 cells were grown on six-well plates to confluency and

transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s




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instructions. At 48 h post-transfection, cells were harvested for qPCR or western blot

analysis.

Cell viability assays

Cell viability was monitored using a Cell Proliferation Reagent Kit I (MTT) (Roche

Applied Science). The SPC-A1, H1299, PC9 or A549 cells transfected with si-ANRIL

(3000 cells/well) were grown in 96-well plates. Cell viability was assessed every 24 h

following the manufacturer’s protocol. All experiments were performed in quadruplicate.

For each treatment group wells were assessed in triplicate.

Flow cytometry

SPC-A1, H1299 or PC9 cells transfected with si-ANRIL were harvested 48 hr after

transfection by trypsinization. After the double staining with FITC-Annexin V and

Propidium iodide (PI) was done using the FITC Annexin V Apoptosis Detection Kit (BD

Biosciences) according to the manufacturer’s recommendations, the cells were analyzed

with a flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software

(BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic

cells, and apoptotic cells, and then the relative ratio of early apoptotic cells were

compared to control transfectant from each experiment. Cells for cell cycle analysis were

stained with PI using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences)

following the protocol and analyzed by FACScan. The percentage of the cells in G0/G1, S,

and G2/M phase were counted and compared.

Tumor formation assay in a nude mouse model




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Female athymic BALB/c nude mice (4-weeks-old) were maintained under pathogen-free

conditions and manipulated according to protocols approved by the Shanghai Medical

Experimental Animal Care Commission. PC9 cells were stably transfected with sh-ANRIL

and empty vector and harvested from 6-well cell culture plates, washed with

phosphate-buffered saline (PBS), and re-suspended at a concentration of 1 × 108 cells/ml.

A total of 100 µL of suspended cells was subcutaneously injected into a single side of the

posterior flank of each mouse. Tumor growth was examined every 3 days, and tumor

volumes were calculated using the equation V = 0.5 × D × d2 (V, volume; D, longitudinal

diameter; d, latitudinal diameter). At 18 days post-injection, mice were euthanized, and

the subcutaneous growth of each tumor was examined. This study was carried out in strict

accordance with the recommendations in the Guide for the Care and Use of Laboratory

Animals of the National Institutes of Health. The protocol was approved by the Committee

on the Ethics of Animal Experiments of the Nanjing medical University.

RNA immunoprecipitation

For immunoprecipitation (IP) of endogenous PRC2 complexes from whole-cell extracts,

cells were lysed. The supernatants were incubated with protein A Sepharose beads

coated with antibodies that recognized EZH2, SNRNP70 or with control IgG (millipore) for

6hr at 4℃. After the beads were washed with wash buffer, the complexes were incubated

with 0.1% SDS/0.5 mg/ml Proteinase K (30 min at 55℃) to remove proteins, respectively.

The PRC2 isolated from the IP materials was further assessed by qPCR analysis (19).

Chromatin Immunoprecipitation




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PC9 cells were treated with formaldehyde and incubated for 10 mins to generate

DNA-protein cross-links. Cell lysates were then sonicated to generate chromatin

fragments of 200-300 bp and immunoprecipitated with EZH2 and H3K27me3-specific

antibody (CST) or IgG as control. Precipitated chromatin DNA was recovered and

analyzed by qPCR.

Western blot assay and antibodies

Cells protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis

(SDS-PAGE), transferred to 0.22μm NC membranes （Sigma）and incubated with specific

antibodies. ECL chromogenic substrate was used to were quantified by densitometry

(Quantity One software; Bio-Rad). GAPDH antibody was used as control, Anti-P21, CDK2,

CDK4, CDK6, P15 and PARP (1:1000) were purchased from Cell Signaling Technology,

Inc (CST); Anti-KLF2 were purchased from sigma.

Statistical analysis

All statistical analyses were performed using SPSS 17.0 software (IBM, Chicago, IL, USA).

The significance of differences between groups was estimated by the Student t-test,

Wilcoxon test or χ2 test. DFS and OS rates were calculated by the Kaplan–Meier method

with the log-rank test applied for comparison. The date of survival were evaluated by

univariate and multivariate Cox proportional hazards models. Variables with p < 0.05 in

univariate analysis were used in subsequent multivariate analysis on the basis of Cox

regression analyses. Kendall’s Tau-b and Pearson correlation analyses were used to

investigate the correlation between ANRIL and KLF2 expressions. Two-sided p-values

were calculated, and a probability level of 0.05 was chosen for statistical significance.




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Results

ANRIL expression was up-regulated and correlated with poor prognosis of NSCLC.

ANRIL expression levels were investigated in 68 paired NSCLC samples and

adjacent histologically normal tissues using qPCR assays. ANRIL expression was

significantly up-regulated (Fold change >1.5, P < 0.01) in 76% (52/68) of cancerous

tissues compared with normal tissues (Fig.1A); the ANRIL expression level in each patient

was shown in Supplementary table S2. Increased ANRIL expression levels in NSCLC

were significantly correlated with tumor size (p = 0.001), and advanced pathological stage

(p=0.007). However, ANRIL expression was not associated with other parameters such

as gender (p = 0.625) and age (p = 0.627) in NSCLC (Table1).

To investigate whether up-regulation of ANRIL is caused by DNA copy number

variation, we referred to array comparative genomic hybridization (aCGH) database in

GSE20393, where deposits 52 lung cancer copy number alteration data generated by

Agilent Human Genome CGH 244A Microarrays. We investigated 26 probes representing

region of ANRIL and extracted GLAD segmented copy number of these probes. The

results showed that there is no significant gain of DNA copy number in this region,

suggesting that up-regulation of ANRIL in lung cancer is not due to copy number variation

(Supplementary Fig. S1A).

Association of ANRIL expression with patients survival

Kaplan-Meier survival analysis was conducted to investigate the correlation between

ANRIL expression and NSCLC patient prognosis. According to relative ANRIL expression

in tumor tissues, the 68 NSCLC patients were classified into two groups: the high ANRIL




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group (n = 34, fold-change ≤ mean ratio); and the low ANRIL group (n = 34, fold-change

≥mean ratio) (Fig. 1B). With respect to progression-free survival (PFS), this was 35.3%

for the high ANRIL group, and 13.6% for the low ANRIL group. Median survival time for

the high ANRIL group was 31 months, and 14 months for the low ANRIL group (Fig. 1C).

The overall survival rate over 3 years for the high ANRIL group was 44.4%, and 20.8% for

the low ANRIL group. Median survival time for the high ANRIL group was 32 months, and

18 months for the low ANRIL group (Fig. 1D).

Univariate analysis identified three prognostic factors: lymph node metastasis; TNM

stage; and ANRIL expression level. Other clinicopathological features such as gender and

age were not statistically significant prognosis factors (Supplementary Table S3).

Multivariate analysis of the three prognosis factors confirmed that HR for ARAIL

expression is 3.509 (95%CI: 1.619-7.607) of progression-free survival, indicating that

ANRIL expression may serve as a potential independent prognostic value in NSCLC

(Supplementary Table S4).

Modulation of ANRIL expression in NSCLC cells

We next performed qPCR analysis to examine the expression of ANRIL in 6 human

NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes

(Fig. 2A). To investigate the functional effects of ANRIL in NSCLC cells, we modulated its

expression through RNA interference. QPCR analysis of ANRIL levels was performed 48

h post-transfection. ANRIL expression was knocked down by 74% in SPC-A1 cells, 75%

in H1299 cells and 94% in PC9 cells by si-ANRIL transfection when compared with control

cells (si-NC) (Fig. 2B).




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Knockdown of ANRIL impaired NSCLC cells proliferation and induced apoptosis

To assess the role of ANRIL in NSCLC, we investigated the effect of targeted

knockdown of ANRIL on cell proliferation. MTT assays revealed that cell growth was

inhibited in SPC-A1, H1299 and PC9 cells transient transfected with si-ANRIL compared

with controls (Fig. 2C). Meanwhile, knockdown of ANRIL expression could also inhibit

A549 cells (with relative low endogenous ANRIL expression level) proliferation

(Supplementary Fig. S1B). Colony formation assay results revealed that clonogenic

survival was inhibited following down-regulation of ANRIL in SPC-A1, H1299 and

PC9cells (Fig. 2D). However, over-expression of ANRIL in 16HBE cells showed no

significant effect on cell proliferation (Supplementary Fig. S1C).

To further examine whether the effect of knockdown ANRIL on proliferation of

NSCLC cells reflected cell cycle arrest, cell cycle progression was analyzed by flow

cytometry analysis. The results revealed that SPC-A1 and PC9 cells transfected with

si-ANRIL had an obvious cell cycle arrest at the G1/G0 phase and had a decreased G2/S

phase (Fig. 3A). To determine whether NSCLC cell proliferation was influenced by cell

apoptosis, we performed flow cytometry and Tunel staining analysis. The results showed

that NSCLC cells transfected with ANRIL siRNA promoted apoptosis in comparison with

that in control cells (Fig. 3B and 2C). These data indicate that ANRIL could promote the

proliferation phenotype of NSCLC cells.

Decreased ANRIL expression inhibits NSCLC cells migration

To investigate the effect of ANRIL knockdown on NSCLC cells migration, transwells

assays were performed. The results showed that decreased ANRIL expression levels




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impeded the migration of SPC-A1 and PC9 cells compared with controls (Supplementary

Fig. S1D).

Down-regulation of ANRIL inhibits NSCLC cells tumorigenesis in vivo.

To explore whether the level of ANRIL expression could affect tumorigenesis, PC9 cells

stably transfected with sh-ANRIL or empty vector were inoculated into female nude mice.

Eighteen days after the injection, the tumors formed in the sh-ANRIL group were

substantially smaller than those in the control group (Fig. 4A). Moreover, the mean tumor

weight at the end of the experiment was markedly lower in the sh-ANRIL group (0.62 ±

0.35 g) compared with the empty vector group (1.41± 0.57 g) (Fig. 4B). QPCR analysis

found that the levels of ANRIL expression in tumor tissues formed from sh-ANRIL cells

were lower than in tumors formed in the control group (Fig. 4C). Tumors formed from

sh-ANRIL-transfected PC9 cells exhibited decreased positive for Ki67 than those from

control cells (Fig. 4D). These findings indicate that knockdown of ANRIL inhibits tumor

growth in vivo.

ANRIL silences KLF2 and P21 transcription by binding with EZH2

Previously studies have indicated that ANRIL could silence p15INK4

transcription and

contribute to cancer cells proliferation (18). The results of qPCR showed that p15 and p16

expression was increased in SPCA1 and H1299 cells with transfection of si-ANRIL;

however, there was no significant difference of p15 expression in PC9 cells when

knockdown of ANRIL expression (Fig. 5A and Supplementary Fig. S1E). There are

evidence showed that EZH2 could regulate KLF2 and P21 expression (20, 21), and our

qPCR results also showed that inhibition of ANRIL expression led to increased KLF2 and




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P21 expression. Moreover, knockdown of EZH2 or SUZ12 could also up-regulate KLF2

and P21 expression (Fig. 5B). Meanwhile, the western blot assays showed the same

results (Fig. 5C), which indicated that KLF2 and P21 could be ANRIL novel targets in PC9

cells. Additionally, we found that ANRIL RNAs were mostly located in the nucleus (Fig.

5D).

To further investigate whether ANRIL repress KLF2 and P21 expression through

binding PRC2, we performed RIP analysis and the results showed that ANRIL could

directly bind with EZH2 in PC9 cells (Fig. 6A). Furthermore, the results of ChIP assays

showed that EZH2 could directly bind to KLF2 and P21 promoter region and mediate

H3K27me3 modification (Fig. 6B). However, knockdown of ANRIL reduced EZH2 binding

with KLF2 and P21 promoter (Fig. 6C). Finally, we detected the KLF2 expression in

NSCLC tissues, and found that there is an inverse relationship between ANRIL and KLF2

expression (Fig. 6D). These data suggested that ANRIL promotes NSCLC PC9 cells

proliferation is not dependent on regulation p15 expression, but also through silencing of

KLF2 and P21 transcription.

Silence of KLF2 is potentially involved in the oncogenic function of ANRIL.

To investigate whether KLF2 is involved in the ANRIL-induced increase in NSCLC

cell proliferation, we performed gain of function assays. The results of western blot

showed that KLF2 expression was significantly up-regulated in PC9 cells transfected with

pCDNA-KLF2 compared with control cells (Fig. 7A). Meanwhile, MTT and colony

formation assay results revealed that over-expression of KLF2 could impaired NSCLC

cells proliferation (Fig. 7B). Moreover, flow cytometry analysis indicated that increased




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KLF2 expression could induce NSCLC cells apoptosis (Fig. 7C). Furthermore, to

determine whether ANRIL regulate NSCLC cell proliferation via repressing KLF2

expression, rescue assays were performed. PC9 cells were co-transfected with si-ANRIL

and si-KLF2, and this was shown to rescue the decreased expression of ANRIL induced

by knockdown of KLF2 (Fig. 7D). The results of MTT and colony formation assay results

indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in PC

cells (Fig. 7E). These data indicate that ANRIL promotes NSCLC cell proliferation through

the down-regulation of KLF2 expression.

Discussion

Recently, numerous pieces of evidence show that many lncRNAs are characterized

and play important roles in cancer pathogenesis, suggesting that they could provide new

insights into the biology of this disease. For example, increased lncRNA HOTTIP is

associated with progression and predicts outcome in hepatocellular carcinoma patients by

regulating HOXA13 expression (22). However, the roles of lncRNAs in NSCLC are still not

well documented, and one of these lncRNAs is metastasis-associated lung

adenocarcinoma transcript 1 (MALAT1), which is a highly conserved nuclear lncRNA and

a predictive marker for metastasis development in lung cancer (23). In our previous

studies, we found that increased lncRNA HOTAIR promoted NSCLC cells invasion and

metastasis via regulating HOXA5 expression, and lncRNA BANCR over-expression could

impaired NSCLC cells proliferation and metastasis by affecting epithelial-mesenchymal

transition (24, 25).




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In this study, we demonstrated that the expression of another lncRNA, ANRIL, is

significantly up-regulated in NSCLC tissues. Specifically, increased ANRIL expression

appears to be a significant, independent predictive value for NSCLC patients. Moreover,

knockdown of ANRIL expression led to the significant inhibition of cell proliferation and the

promotion of apoptosis both in vitro and in vivo. These findings suggest that ANRIL plays

a direct role in the modulation of cell proliferation and NSCLC progression, and could be a

useful novel prognostic or progression marker for NSCLC. As more and more lncRNAs

are studied, many have been shown to function by binding with PRC2 and silencing

downstream target genes that involved in multiple cancers including NSCLC (26, 27).

ANRIL has been reported to involve in cancer cells proliferation by silencing p15INK4

expression. In this study, we found that ANRIL is mostly located in cell nucleus and could

directly bind with EZH2, an core subunit of PRC2, resulted in repressing KLF2 and P21

transcription. However, knockdown of ANRIL could not influence p15INK4

expression in

NSCLC PC9 cells, which indicated that ANRIL contributed to NSCLC cell proliferation is

not dependent on regulating p15INK4

, but also could through silencing KLF2 and P21

transcription.

The Kruppel-like factor (KLF) family transcription factors, with Cys2/His2 zinc-finger

domains, could function as suppressors or activators in a cell type and

promoter-dependent manner and involve in cell differentiation and proliferation (28, 29).

Some KLF members are emerging as tumor suppressor genes due to their roles in the

inhibition of proliferation, migration, and induction of apoptosis (30, 31). KLF2, as an

member of KLF family, is diminished in many cancers and possesses tumor-suppressor




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features such as inhibition of cell proliferation mediated by KRAS (32-34). Moreover, there

is evidence showed that EZH2 could silence KLF2 expression and block the

tumor-suppressor features of KLF2, which is partly mediated by p21 (21). Our results also

showed lncRNA ANRIL take part in NSCLC cells proliferation by epigenetic silencing

KLF2 and P21 transcription, and KLF2 inactivation further led to the decreased P21

expression. As more and more studies indicated that lncRNAs are often expressed in a

spatial- or temporal-specific pattern, and more cell and tissue -specific pattern. Our results

also showed that even in NSCLC cells, lncRNA ANRIL could regulate different target

genes in different cell lines, which suggested that lncRNA, especially ANRIL, can

influence the same cell biological function via regulating differnet target genes dependent

on different cell lines.

To date, although only a small number of lncRNAs have been well characterized, they

have been shown to regulate gene expression at various levels, including chromatin

modification and post-transcriptional processing (35, 36). Here, the possible other targets

and mechanism that underlie such regulatory behaviors still remain to be fully understood

despite our observation of ANRIL-induced NSCLC cell proliferation. In summary, the

expression of ANRIL was significantly increased in NSCLC tissues, suggesting that its

up-regulation may be a negative prognostic factor for NSCLC patients, indicative of poor

survival rates, and a higher risk for cancer metastasis. We showed that ANRIL possibly

regulates the proliferation ability of NSCLC cells, partially through its regulation of the

KLF2 and P21, which indicated that lncRNAs contribute to different cancer cells biological

function maybe through regulating different target genes. Our findings further the




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understanding of NSCLC pathogenesis, and facilitate the development of

lncRNA-directed diagnostics and therapeutics against cancers.

Acknowledgements

We are very grateful to professor Xiongbin Lu for providing the ANRIL over-expression

plamid.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Reference 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11-30.

2. Spira A, Ettinger DS. Multidisciplinary management of lung cancer. N Engl J Med.

2004;350:379-92.

3. Wistuba, II. Genetics of preneoplasia: lessons from lung cancer. Curr Mol Med. 2007;7:3-14.

4. Verdecchia A, Francisci S, Brenner H, Gatta G, Micheli A, Mangone L, et al. Recent cancer

survival in Europe: a 2000-02 period analysis of EUROCARE-4 data. Lancet Oncol. 2007;8:784-96.

5. Finishing the euchromatic sequence of the human genome. Nature. 2004;431:931-45.

6. Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T, Mortazavi A, et al. Landscape of

transcription in human cells. Nature. 2012;489:101-8.

7. Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7

catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression.

Genome Res. 2012;22:1775-89.

8. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281-97.

9. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development.

Nat Rev Genet. 2014;15:7-21.

10. Geisler S, Coller J. RNA in unexpected places: long non-coding RNA functions in diverse cellular

contexts. Nat Rev Mol Cell Biol. 2013;14:699-712.

11. Loewer S, Cabili MN, Guttman M, Loh YH, Thomas K, Park IH, et al. Large intergenic

non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells. Nat Genet.

2010;42:1113-7.

12. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev

Genet. 2009;10:155-9.

13. Wapinski O, Chang HY. Long noncoding RNAs and human disease. Trends Cell Biol.

2011;21:354-61.

14. Tsai MC, Spitale RC, Chang HY. Long intergenic noncoding RNAs: new links in cancer

progression. Cancer Res. 2011;71:3-7.

15. Spizzo R, Almeida MI, Colombatti A, Calin GA. Long non-coding RNAs and cancer: a new

frontier of translational research? Oncogene. 2012;31:4577-87.

16. Zhang EB, Kong R, Yin DD, You LH, Sun M, Han L, et al. Long noncoding RNA ANRIL



http://www.ncbi.nlm.nih.gov/pubmed/?term=Lu%20X%5Bauth%5D


19

indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of

miR-99a/miR-449a. Oncotarget. 2014;5:2276-92.

17. Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, et al. Long non-coding RNA

ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene.

Oncogene. 2011;30:1956-62.

18. Chen D, Zhang Z, Mao C, Zhou Y, Yu L, Yin Y, et al. ANRIL inhibits p15 through the TGFbeta1

signaling pathway in human esophageal squamous cell carcinoma. Cell Immunol. 2014;289:91-6.

19. Yoon JH, Abdelmohsen K, Srikantan S, Yang X, Martindale JL, De S, et al. LincRNA-p21

suppresses target mRNA translation. Mol Cell. 2012;47:648-55.

20. Seward S, Semaan A, Qazi AM, Gruzdyn OV, Chamala S, Bryant CC, et al. EZH2 blockade by

RNA interference inhibits growth of ovarian cancer by facilitating re-expression of p21(waf1/cip1) and

by inhibiting mutant p53. Cancer Lett. 2013;336:53-60.

21. Taniguchi H, Jacinto FV, Villanueva A, Fernandez AF, Yamamoto H, Carmona FJ, et al. Silencing

of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer. Oncogene.

2012;31:1988-94.

22. Quagliata L, Matter MS, Piscuoglio S, Arabi L, Ruiz C, Procino A, et al. Long noncoding RNA

HOTTIP/HOXA13 expression is associated with disease progression and predicts outcome in

hepatocellular carcinoma patients. Hepatology. 2014;59:911-23.

23. Gutschner T, Hammerle M, Eissmann M, Hsu J, Kim Y, Hung G, et al. The noncoding RNA

MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells. Cancer Res.

2013;73:1180-9.

24. Liu XH, Liu ZL, Sun M, Liu J, Wang ZX, De W. The long non-coding RNA HOTAIR indicates a

poor prognosis and promotes metastasis in non-small cell lung cancer. BMC Cancer. 2013;13:464.

25. Sun M, Liu XH, Wang KM, Nie FQ, Kong R, Yang JS, et al. Downregulation of BRAF activated

non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes

metastasis by affecting epithelial-mesenchymal transition. Mol Cancer. 2014;13:68.

26. Beckedorff FC, Ayupe AC, Crocci-Souza R, Amaral MS, Nakaya HI, Soltys DT, et al. The

intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the

expression of RASSF1A and increasing cell proliferation. PLoS Genet. 2013;9:e1003705.

27. Brockdorff N. Noncoding RNA and Polycomb recruitment. RNA. 2013;19:429-42.

28. Black AR, Black JD, Azizkhan-Clifford J. Sp1 and kruppel-like factor family of transcription

factors in cell growth regulation and cancer. J Cell Physiol. 2001;188:143-60.

29. Kaczynski J, Cook T, Urrutia R. Sp1- and Kruppel-like transcription factors. Genome Biol.

2003;4:206.

30. Shen P, Sun J, Xu G, Zhang L, Yang Z, Xia S, et al. KLF9, a transcription factor induced in

flutamide-caused cell apoptosis, inhibits AKT activation and suppresses tumor growth of prostate

cancer cells. Prostate. 2014;74:946-58.

31. Hsu LS, Chan CP, Chen CJ, Lin SH, Lai MT, Hsu JD, et al. Decreased Kruppel-like factor 4

(KLF4) expression may correlate with poor survival in gastric adenocarcinoma. Med Oncol.

2013;30:632.

32. Wu J, Lingrel JB. KLF2 inhibits Jurkat T leukemia cell growth via upregulation of

cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene. 2004;23:8088-96.

33. Wang F, Zhu Y, Huang Y, McAvoy S, Johnson WB, Cheung TH, et al. Transcriptional repression

of WEE1 by Kruppel-like factor 2 is involved in DNA damage-induced apoptosis. Oncogene.




20

2005;24:3875-85.

34. Fernandez-Zapico ME, Lomberk GA, Tsuji S, DeMars CJ, Bardsley MR, Lin YH, et al. A

functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of

KRAS-mediated cell growth. Biochem J. 2011;435:529-37.

35. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell.

2009;136:629-41.

36. Nagano T, Fraser P. No-nonsense functions for long noncoding RNAs. Cell. 2011;145:178-81.

Table 1 Correlation between ANRIL expression and clinicopathological characteristics of

NSCLC patients

Characteristics ANRIL P

High No. cases

(34)

Low No. cases

(34)

Chi-squared

test P-value

Age(years) 0.627

≤65 17 19

>65 17 15

Gender 0.625

Male 18 20

Female 16 14

Histological subtype 0.324

Squamous cell

carcinoma

22 18

Adenocarcinoma 12 16

TNM Stage 0.007*

Ia + Ib 4 15

IIa + IIb 14 12

IIIa 16 7

Tumor size 0.001*

≤5cm 13 26

>5cm 21 8

Lymph node

metastasis

0.051

Negative 11 19

Positive 23 15

Smoking History 0.793

Smokers 23 24

Never Smokers 11 10

* Overall P<0.05

Figure legends




21

Figure 1. Relative ANRIL expression in NSCLC tissues and its clinical significance.

(A) Relative expression of ANRIL in NSCLC tissues (n = 68) compared with

corresponding non-tumor tissues (n = 68). ANRIL expression was examined by qPCR and

normalized to GAPDH expression. Results were presented as the fold-change in tumor

tissues relative to normal tissues. (B) ANRIL expression was classified into two groups. (C,

D) Kaplan–Meier disease-free survival and overall survival curves according to ANRIL

expression levels. *P < 0.05, **P < 0.01.

Figure 2. Effects of knockdown of ANRIL on NSCLC cell viability and apoptosis in

vitro. (A) ANRIL expression levels of NSCLC cell lines (PC9, SPC-A1, NCI-H1975,

H1299, and H358 and H520) compared with that in normal human bronchial epithelial

cells (16HBE). (B) SPC-A1, H1299 and PC9 cells were transfected with si-ANRIL. (C)

MTT assays were used to determine the cell viability for si-ANRIL-transfected SPC-A1,

H1299 and PC9 cells. Values represented the mean ± s.d. from three independent

experiments. (D) Colony-forming assays were conducted to determine the proliferation of

si-ANRIL-transfected SPC-A1, H1299 and PC9 cells. Flow cytometry assays were

performed to analysis the cell cycle progression and apoptosis when NSCLC cells

transfected with si-ANRIL. *P<0.05, **P<0.01.

Figure 3. Effects of knockdown of ANRIL on NSCLC cell apoptosis in vitro. (A) The

bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated.

(B) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal

apoptotic cells; LR, early apoptotic cells. (C) Apoptosis was determined by Tunel staining.




22

All experiments were performed in biological triplicates with three technical

replicates.*P<0.05, **P<0.01.

Figure 4. Effects of down-regulation of ANRIL on tumor growth in vivo. (A) The

tumor volume was calculated once every three days after injection of PC9 cells stably

transfected with sh-ANRIL or empty vector. Points, mean (n=7); bars indicate S.D. (B)

Tumor weights are represented as means of tumor weights ± s.d. (C) QPCR analysis of

ANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. (D).

Tumors developed from sh-ANRIL transfected PC9 cells showed lower Ki67 protein levels

than tumors developed by control cells. Upper: H & E staining; Lower: immunostaining.

*P<0.05, **P<0.01 and N.S. not significant.

Figure 5. ANRIL could silence KLF2 and P21 expression. (A) The levels of p15INK4B

,

p21, and KLF2 mRNA were determined by qPCR when SPC-A1 and PC9 cells

transfected with si-ANRIL and results are expressed relative to the corresponding values

for control cells. (B,C) The levels of p21 and KLF2 mRNA and protein levels were

determined by qPCR when PC9 cells transfected with si-EZH2 or si-SUZ12. (D) ANRIL

expression levels in cell cytoplasm or Nucleus of NSCLC cell lines SPC-A1, PC9 and

H520 were detected by qPCR.

Figure 6. ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription.

(A) RIP with rabbit monoclonal anti-EZH2, preimmune IgG or 10% input from PC9 cell

extracts. RNA levels in immunoprecipitates were determined by qPCR. Expression levels

of ANRIL RNA were presented as fold enrichment in EZH2 relative to IgG

immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG




23

immunoprecipitates were used as positive control. (B, C) ChIP–qPCR of EZH2 occupancy

and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control;

ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9

cells treated with ANRIL siRNA (48 h) or scrambled siRNA. (D) Analysis of the

relationship between ANRIL expression and KLF2 mRNA level (△Ct value) in 40 NSCLC

tissues. The mean values and s.d.s were calculated from triplicates of a representative

experiment.

Figure 7. Over-expression of KLF2 expression inhibit PC9 cells proliferation. PC9

cells were transfected with pCDNA-KLF2 or co-transfected with si-ANRIL and si-KLF2. (A)

The protein level of KLF2 in PC9 cell transfected with pCDNA-KLF2 was detected by

western blot. (B) MTT assays and colony-forming assays were used to determine the cell

viability for pCDNA-KLF2-transfected PC9 cells. Values represent the mean ± s.d. from

three independent experiments. (C) Apoptosis was determined by flow cytometry. UL,

necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. (D) The protein level

of KLF2 in PC9 cell co-transfected with si-ANRIL and si-KLF2 was detected by western

blot. (E) MTT assays and colony-forming assays were used to determine the cell viability

for si-ANRIL and si-KLF2 co-transfected PC9 cells. Values represent the mean ± s.d. from

three independent experiments. *P < 0.05 and **P < 0.01

























Published OnlineFirst December 12, 2014.Mol Cancer Ther Feng-qi Nie, Ming Sun, Jin-song Yang, et al. KLF2 and P21 expressioncancer cells proliferation and inhibits apoptosis by silencing Long noncoding RNA ANRIL promotes non small cell lung

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Long non coding RNA ANRIL promotes non small cell lung ......Dec 27, 2014 · 1 Long non-coding RNA ANRIL promotes non small cell lung cancer cells proliferation and . inhibits. apoptosis